Intracellular pH (ph(i)) in squid axons is regulated by an electroneutral ion transport syste, located in the cell membrane, which responds to intracellular acid loads (i.e., decreases in ph(i)) by accumulating HCO3 and Na+ in exchange for Cl- and H+. The stoichiometry is one equivalent of Na+ taken up for each equivalent of Cl- lost, and for each two equivalents of alkali added to or acid removed from the cell. The gradient model predicts that net transport should halt when the sum of gradients of transported ions (i.e., DeltaG(net)) is zero, and should reverse when the sum of gradients is reversed (i.e., DeltaG(net) greater than 0). Preliminary experiments confirm that the net, equivalent HCO3- flux (i.e., the net flux of HCO3- plus the net flux of H+, if any, in the opposite direction, as measured with a pH microelectrode), at least under one set of experimental conditions, is zero when DeltaG(net) = 0, and is reversed when DeltaG(net) greater than 0. I propose to continue testing predictions of the gradient model: (i) With the transporter initially in equilibrium (DeltaG(net) = 0), altering the gradient of any one transported ion should throw the transporter into forward or reverse. (ii) Presumptive reversal of the transporter should be accompanied by an extra net influx of Cl- and extra net efflux of Na+; these fluxes of HCO3-, Na+ and Cl- should be dependent on one another and blocked by the same inhibitors. (iii) The reversed transporter should have the same stoichiometry as the forward transporter. In addition, I will determine whether the purported reversed transporter requires ATP (as does the forward transporter), and whether its rate is influenced by pH(i) at a fixed DeltaG(net). The experiments will be performed on internally dialyzed squid giant axons. Net, equivalent HCO3- fluxes will be determined from rates of ph(i) change, using pH microelectrodes. Net Na+ and Cl- fluxes will be determined from isotopic fluxes. These experiments should provide new insight into the mechanism by which the cell maintains a stable ph(i) and thereby avoids the consequences of intracellular acidosis: deranged cellular metabolism, altered ionic conductances, decreased muscle tension/myocardial contractility, altered [CA++](i), and others.